Deaths due to infectious diseases are on the rise fueled largely by an increase in pathogen drug resistance. Accordingly, there is a critical need for the development of new antibiotics to combat antimicrobial-resistant infections. Historically, bacteria have served as the foremost source of new antibiotic scaffolds demonstrating an incredible capacity for generating antimicrobial compounds. Shockingly, all existing natural-product-based bacterial-derived antibiotics have come from an extremely minor fraction of the Earth's microbial diversity. Specifically, only about 1% of bacteria are currently culturable meaning that all of society's most valuable antibiotic discoveries have originated from a decided minority of bacterial taxa. It has been proposed and substantiated by metagenome studies that the yet uncultured 99% of bacteria harbor an abundance of potential antibiotic substances awaiting discovery. However, current methods for unearthing these compounds from uncultured bacteria pose numerous challenges that severely hamper efforts to effectively mine this resource. Considering these limitations, this proposal poses the novel hypothesis that yet uncultured bacteria lack the genes and gene products that enable culturable microbes to survive and proliferate in the lab. To test this hypothesis, our group is developing an innovative technology for creating culturable chimeric clones by introducing large fragments of genomic DNA from bacteria exhibiting robust growth in the lab into as of yet uncultured bacteria. These proposed studies will be accomplished by means of four specific aims. Aim1 involves carrying out the in situ transfection of uncultured bacteria in soils and sediments with a bacterial artifical chromosome (BAC) library composed of genomic DNA from a readily culturable Gram-positive bacterium and selecting for new (previously uncultured) chimeric clones. Aim 2 is designed to test for the expression of BAC encoded genes, identify how the gene inserts enable growth, and assess microbes for the production of antimicrobial secondary metabolites. Aim 3 will provide two additional BAC libraries containing genomic DNA from a Gram-positive high-GC bacterium and a representative of the largely uncultured, but abundant Acidobacteria for further chimera-generation studies. Aim 4 will test the capacity of this method to generate chimeric clones from marine invertebrate samples for the targeted procurement of uncultured microbial producers of two classes of antiinfective natural products. Several important outcomes are anticipated as a result of this research. Importantly, we will establish a foundation for using this innovative technique to grow previously uncultured bacteria and obtain their bioactive natural products. In addition, we expect that some of the newly accessible compounds will include unusual scaffolds not encountered from existing culturable bacteria. In the long-term, we anticipate that new natural products from previously uncultured bacteria will provide abundant opportunities to procure novel antimicrobials that are desperately needed to control the increasing loss of human life attributable to antibiotic-resistant infections.